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(R)-PFI-2 Is a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity in Cells

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(R)-PFI-2 Is a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity in Cells (R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells Dalia Barsyte-Lovejoya,1,2, Fengling Lia,1, Menno J. Oudhoffb,1, John H. Tatlockc,1, Aiping Donga, Hong Zenga, Hong Wua, Spencer A. Freemand, Matthieu Schapiraa,e, Guillermo A. Senisterraa, Ekaterina Kuznetsovaa, Richard Marcellusf, Abdellah Allali-Hassania, Steven Kennedya, Jean-Philippe Lambertg, Amber L. Couzensg, Ahmed Amanf, Anne-Claude Gingrasg,h, Rima Al-Aware,f, Paul V. Fishi,3, Brian S. Gerstenbergerj, Lee Robertsk, Caroline L. Bennl, Rachel L. Grimleyl, Mitchell J. S. Braamb, Fabio M. V. Rossib,m, Marius Sudoln, Peter J. Browna, Mark E. Bunnagek, Dafydd R. Owenk, Colby Zaphb,o, Masoud Vedadia,e,2, and Cheryl H. Arrowsmitha,p,2 aStructural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7; bBiomedical Research Centre, University of British Columbia, Vancouver, BC, Canada V6T1Z3; cWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, San Diego, CA 92121; dDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T1Z3; eDepartment of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada M5S 1A8; fDrug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON, Canada M5G 0A3; gCentre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada M5G 1X5; hDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; iWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom; jWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340; kWorldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, MA 02139; lNeusentis Research Unit, Pfizer Worldwide Research and Development, Cambridge CB21 6GS, United Kingdom; mDepartment of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V6T1Z3; nWeis Center for Research, Geisinger Clinic, Danville, PA 17821; oDepartment of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6T1Z3; and pPrincess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, ON, Canada M5G 1L7 Edited by Robert J. Fletterick, University of California, San Francisco School of Medicine, San Francisco, CA, and approved July 22, 2014 (received for review April 22, 2014) SET domain containing (lysine methyltransferase) 7 (SETD7) is impli- do not prematurely develop cancer (17, 18). Thus, the exact func- cated in multiple signaling and disease related pathways with a broad tional role of SETD7 in normal or disease biology is still an open diversity of reported substrates. Here, we report the discovery of app question (10). (R)-PFI-2—a first-in-class, potent (Ki = 0.33 nM), selective, and cell- SETD7 contains a SET [Su(var)3–9 and Enhancer of Zeste- — active inhibitor of the methyltransferase activity of human SETD7 Trithorax] domain that is conserved among many S-adeno- S R and its 500-fold less active enantiomer, ( )-PFI-2. ( )-PFI-2 exhibits an sylmethionine (SAM)-dependent protein lysine methyltransferases unusual cofactor-dependent and substrate-competitive inhibitory (PKMTs) (19). SETD7 has robust monomethyltransferase activity mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adeno- Significance sylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition Protein methyltransferases constitute an emerging but under- for binding to endogenous SETD7 in MCF7 cells pretreated with (R)- characterized class of therapeutic targets with diverse roles in PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocop- normal human biology and disease. Small-molecule “chemical ied the effects of Setd7 deficiency on Hippo pathway signaling, via probes” can be powerful tools for the functional characterization modulation of the transcriptional coactivator Yes-associated protein of such enzymes, and here we report the discovery of (R)-PFI-2—a (YAP) and regulation of YAP target genes. In confluent MCF7 cells, first-in-class, potent, highly selective, and cell-active inhibitor of (R)-PFI-2 rapidly altered YAP localization, suggesting continuous the methyltransferase activity of SETD7 [SET domain containing and dynamic regulation of YAP by the methyltransferase activity (lysine methyltransferase) 7]—and two related compounds for of SETD7. These data establish (R)-PFI-2 and related compounds as control and chemoproteomics studies. We used these com- MEDICAL SCIENCES a valuable tool-kit for the study of the diverse roles of SETD7 in pounds to characterize the role of SETD7 in signaling, in the cells and further validate protein methyltransferases as a drug- Hippo pathway, that controls cell growth and organ size. Our gable target class. work establishes a chemical biology tool kit for the study of the diverse roles of SETD7 in cells and further validates protein epigenetics | chemical biology | chemical probe methyltransferases as a druggable target class. Author contributions: D.B.-L., M.J.O., J.H.T., M. Schapira, J.-P.L., A.L.C., A.-C.G., R.A.-A., P.V.F., rotein methyltransferases play diverse roles in the epigenetic B.S.G., L.R., C.L.B., R.L.G., P.J.B., M.E.B., D.R.O., C.Z., M.V., and C.H.A. designed research; Pregulation of gene transcription, silencing, chromatin struc- D.B.-L., F.L., M.J.O., A.D., H.Z., H.W., S.A.F., G.A.S., E.K., R.M., A.A.-H., S.K., J.-P.L., A.L.C., ture establishment, maintenance, DNA repair, and replication. SET A.A., and R.L.G. performed research; M.J.S.B., F.M.V.R., and M. Sudol contributed new domain containing (lysine methyltransferase) 7 (SETD7) (SET9; reagents/analytic tools; D.B.-L., F.L., M.J.O., J.H.T., A.D., H.W., M. Schapira, E.K., R.M., A.A.-H., S.K., J.-P.L., A.L.C., A.A., R.A.-A., P.V.F., B.S.G., L.R., C.L.B., R.L.G., D.R.O., C.Z., M.V., and C.H.A. SET7/9, KMT7) was one of the first protein lysine methyltrans- analyzed data; and D.B.-L., M.J.O., J.H.T., M. Schapira, A.-C.G., P.J.B., M.E.B., D.R.O., C.Z., ferases to be discovered and was originally characterized as a mon- M.V., and C.H.A. wrote the paper. omethyltransferase of lysine 4 on histone H3 (H3K4me1) (1, 2). The authors declare no conflict of interest. Subsequently, SETD7 has been shown to have a very broad target This article is a PNAS Direct Submission. specificity in vitro, including transcriptional regulators such as – Data deposition: The atomic coordinates and structure factors have been deposited in the TAF10,p53,ER,p65,STAT3,Rb,Mypt,Tat,andFoxo3(316). Protein Data Bank, www.pdb.org (PDB ID code 4JLG). SETD7 is also reported to regulate DNA methyltransferase 1 1D.B.-L., F.L., M.J.O., and J.H.T. contributed equally to this work. (DNMT1) in a brain-specific manner (14) and functionally interacts 2 β To whom correspondence may be addressed. Email: [email protected], m.vedadi@ with Pdx1 in pancreatic cells (6). The diverse nature of its sub- utoronto.ca, or [email protected]. strates has implicated SETD7 in multiple molecular pathways in- 3Present address: University College London School of Pharmacy, London WC1N 1AX, volved in cancer, metabolism, and inflammation. Despite these United Kingdom. diverse substrates and molecular pathways, mice with a genetic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deletion of Setd7 have no obvious developmental deficiencies and 1073/pnas.1407358111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407358111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12853–12858 Downloaded by guest on September 24, 2021 investigation of the functional biology of these enzymes, as well as the exploration of their potential as therapeutic targets (25). Here, we describe the discovery of (R)-PFI-2, a potent and se- lective inhibitor of SETD7, and its use in modulation of the Hippo pathway by SETD7 inhibition. Thus, (R)-PFI-2, together with its 500-fold less-active enantiomer (S)-PFI-2 and a bio- tinylated derivative of (R)-PFI-2, provides a chemical probe tool kit to interrogate the biology of SETD7. Results (R)-PFI-2 Is a Potent, Selective Inhibitor of SETD7 Methyltransferase Activity. The discovery of (R)-PFI-2 (Fig. 1A) was initiated with a high-throughput screen (HTS) of a 150,000-compound subset of Pfizer’s full HTS collection in an in vitro enzymatic assay of recombinant SETD7. After several rounds of structure-guided molecular design, including optimization of cell permeability, the inhibition of SETD7 in vitro catalytic activity was improved >100-fold compared with the initial HTS hit (IC50 of 2.1 μM), resulting in (R)-PFI-2 (synthesis provided in SI Appendix). A robust radioactivity-based assay was used to determine kinetic parameters for methyltransferase activity of SETD7 and to char- Fig. 1. (R)-PFI-2 is a potent inhibitor of SETD7. (A) Chemical structures of acterize inhibitors (Table 1 and SI Appendix,Fig.S1). (R)-PFI-2 SETD7 inhibitors (R)-PFI-2 and its less-active enantiomer (S)-PFI-2. (B) The inhibited the methyltransferase activity of human SETD7 with an effect of (R)-PFI-2 (●) and (S)-PFI-2 (▲) on methyltransferase activity of IC50 value of 2.0 ± 0.2 nM whereas its enantiomer, (S)-PFI-2, was ± ± μ SETD7. Compounds inhibited SETD7 activity with IC50 values of 2.0 0.2 nM 500-fold less active (IC50 value of 1.0 0.1 M), making the latter (Hill slope, 0.8) and 1.0 ± 0.1 μM (Hill slope: 0.7), respectively.
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